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Assembly of tobacco mosaic virus and TMV-like pseudovirus particles in Escherichia coli

  • D.-J. Hwang
  • I. M. Roberts
  • T. M. A. Wilson
Conference paper
Part of the Archives of Virology Supplementum book series (ARCHIVES SUPPL, volume 9)

Summary

High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli. Native tobacco mosaic virus (TMV) CP expressed in E. coli remains soluble but has a non-acetylated N-terminal Ser residue and, following extraction, is unable to package TMV RNA in vitro under standard assembly conditions. Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV. All TMV CPs made in E. coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP. TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4kb ssRNA genome. Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence. Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles. When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2–18h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4µg/mg total bacterial protein). In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E. coli system also offers a rapid approach for studying the molecular requirements for plant virus “self-assembly” in vivo. Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.

Keywords

Coat Protein Tobacco Mosaic Virus Coat Protein Gene Rabbit Polyclonal Antiserum Tobacco Mosaic Virus Coat Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • D.-J. Hwang
    • 1
  • I. M. Roberts
    • 2
  • T. M. A. Wilson
    • 2
  1. 1.AgBiotech CenterCook College, Rutgers UniversityNew BrunswickUSA
  2. 2.Department of VirologySCRIInvergowrie, DundeeScotland

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